Erythropoietin (EPO) is a 165 amino acid, 34 kilodalton (kDa) glycoprotein hormone which is the principal factor responsible for the regulation of red blood cell production during steady-state conditions and for accelerating recovery of red blood cell mass following hemorrhage. The primary site for EPO synthesis in adult organisms is the kidney. The liver synthesizes lower levels of EPO, and some evidence suggests that there is an additional contribution from macrophages in the bone marrow. The primary stimulus for increased EPO synthesis is tissue hypoxia, which results from decreased blood O2 availability [Jelkmann, W., Physiol. Reviews, 72: 449 (1992)]. The principal function of EPO is to act in concert with other growth factors to stimulate the proliferation and maturation of responsive bone marrow erythroid precursor cells.
A gene for a human EPO receptor (EPO-R) has been isolated and mapped to the p region of chromosome 19 [Winkelman, J. C. et al., Blood, 76: 24 (1990)]. cDNA analysis predicts this receptor to be a 55 kDa, 508 amino acid residue transmembrane protein comprised of a 24 amino acid signal peptide, a 226 amino acid external segment, a 22 amino acid transmembrane segment, and a 236 amino acid cytoplasmic domain [Youssoufian, H. et al., Blood, 81: 2223 (1993)]. The properties of this receptor, including the presence of a set of four conserved cysteine residues and a WSXWS motif in the external segment, place it in the hematopoietin/cytokine superfamily of receptors that also includes the receptors for interelukins IL-3, IL-4, IL-6, IL-7, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), the beta-subunit of the IL-2 receptor and others [Cosman, D., Cytokine 5:95 (1993)]. Cells known to express EPO-R include megakaryocytes, erythoid progenitors, endothelial cells, and, possibly, neurons [Landschulz, K. T. et al., Blood 73: 1476 (1989); Youssoufian, H. et al., Blood 81: 2223 (1993); Fraser, J. K. et al., Exp. Hematol., 17: 10 (1989); Anagnostou, A. et al., Proc. Natl. Acad. Sci. USA, 91: 3974 (1994); and Digicaylioglu, M. et al., Proc. Natl. Acad. Sci. USA, 92: 3717 (1995)].
Ligand binding studies demonstrate the existence of distinct high (Kd=75-100 pM) and low (Kd=220-800 pM) affinity receptors for EPO [Broudy, V. C. et al., Blood, 77: 2583 (1991); Harris, K. W. et al., J. Biol. Chem., 267: 15205 (1992); and Landschulz, K. T. et al., Blood, 73: 1476 (1989)], and cross-linking studies show the presence of multiple cross-linked species [Miura, O. and J. Ihle, Blood, 81: 1739 (1993)]. EPO-R dimerization and janus kinase 2 (JAK2) activation are considered to be first steps in the signal transduction process [Watowich, S. S. et al., Proc. Natl. Acad. Sci. USA, 89: 2140 (1992); Witthuhn, B. A. et al., Cell, 74: 227 (1993); and Tanner, J. W. et al., J. Biol. Chem., 270: 6523 (1995)].
Although the details of the interactions of the components of the EPO receptor complex and the mechanism of signal transduction by this complex are not yet fully understood, x-ray crystallography studies suggest that the EPO-R when not bound to a ligand (i.e., “unliganded”) exists as a dimer in an open-scissors-like conformation with the C-terminal end of the subdomain 2 regions being over 70 angstroms apart [Livnah, O. et al., Science, 283: 987 (1999)]. In the ligand bound EPO-R/EPO structure (i.e., “liganded EPO-R/EPO”), these C-terminal regions become much closer (˜30 angstroms). Thus, it is envisioned that the preformed EPO-R dimer, by keeping the cytoplasmic domains apart, is in an inactivated state, but ligand occupancy brings the extracellular and cytoplasmic domains into proximity to allow signaling. Fragment complementation assays confirmed these data by demonstrating a dramatic ligand-induced enhancement of proximity of the cytoplasmic domain of EPO-R dimers [Remy, I. et al., Science, 283: 990 (1999)]. Together, these studies implicate the existence of preformed EPO-R dimers that are activated by a distinct conformational change in response to ligand.
Within the erythroid lineage, EPO seems to act in concert with other growth factors such as stem cell factor (SCF), insulin-like growth factor-I (IGF-I), and interleukin-3 (IL-3) to ensure the expansion and maturation of immature erythrocytes [Muta, K. et al., J. Clin. Invest., 94: 34 (1994)]. In particular, EPO has been found to interrupt the normal apoptotic cycle experienced by erythroid progenitors as they progress from erythrocyte colony forming units (CFU-E) through the basophilic erythroblast stage [Koury, M. J. and M. C. Bondurant, Science, 248: 378 (1990); and Nijhof, W. et al., Exp. Hematol., 23: 369 (1995)].
In conjunction with IL-3, EPO also seems to have an effect on the earliest erythroid precursor, the erythrocyte burst forming unit (BFU-E), which gives rise to CFU-E. In this case, evidence suggests its activities are not limited to maintaining cell viability. Both IL-3 and EPO are reported to induce proliferation of BFU-E, but only EPO seems capable of initiating differentiation/maturation of BFU-E [Carroll, M. et al., Proc. Natl. Acad. Sci. USA, 92: 2869 (1995); Liboi, E. et al., Proc. Natl. Acad. Sci. USA, 90: 11351 (1993); Krosl, J. et al., Blood, 85: 50 (1995); and Dai, C. H. et al., Blood, 78: 2493 (1991)].
Deficient (or inefficient) EPO production relative to hemoglobin level is associated with certain forms of anemia. These include anemia of renal failure and end-stage renal disease [Kurtz, A. and Eckardt, K-U., Contrib. Nephrol., 87: 15 (1990)], anemia of chronic disorders (chronic infections and rheumatoid arthritis) [Means, R. T., Stem Cells, 13: 32 (1995)], autoimmune diseases [Jelkmann, W., Physiol. Reviews, 72: 449 (1992)], AIDS [Doweiko, J. P., Blood Reviews, 7:121 (1993)], and malignancy [Miller, C. B. et al., New Engl. J. Med., 322: 1689 (1990)]. Many of these conditions are associated with the generation of inerleukin-1 (IL-1) and a factor that has been shown to be an inhibitor of EPO activity [Jelkman, W. E. et al., Ann. NY Acad. Sci., 718: 300 (1994); and Jelkman, W. et al., Life Sci., 50: 301 (1991)].
At present, the primary treatment for anemia induced by these conditions is the administration of recombinant EPO via subcutaneous or intravenous injection. While the use of recombinant EPO has significantly improved the quality of life of these patients, there are some hardships associated with EPO treatment in that chronic treatment requires repeated administration by injection, which is both inconvenient and costly for the patient. Thus, the discovery of effective and safe orally active small molecular EPO mimetics has the potential to enhance the standard of therapy beyond the current recombinant EPO therapy.